Transgenic non-human mammal with an oncogenic mutant of the c-Raf-1 gene

ABSTRACT

The invention relates to a transgenic non-human mammal whose cells express a constitutively active oncogenic mutantof the kinase-domain of the Raf-1 gene or a protein coded by a corresponding normal allele or derivative of the A, B, or c-Raf-1 gene.

FIELD OF THE INVENTION

[0001] The invention relates to a transgenic non-human mammal, to amethod for producing the latter, to the utilization thereof, to a celltissue therefrom, to a method for producing such cell tissue, to theutilization thereof, to a recombinant DNA expression vector and to theutilization of such vector.—The term non-human mammal refers totaxonomically higher units than animal species. Transgenic animals areorganisms carrying an additional gene not originating from theirspecies, that is a foreign gene in their genome. For the purpose of theinvention in particular, such transgenic animals are meant that have theforeign gene also in the germ cells, that is which hand on the foreigngene vertically, i.e. from generation to generation. If a specialtransgenic animal has been created, further corresponding transgenicanimals may be obtained by breeding. Transgenic animals are known in theart in various embodiments, and various methods for producing transgenicanimals are also known. As an example only, reference is made todocument R. Jaenisch, Science, Vol. 240, 10, 1988, page 1468 ff., andthe documents cited therein. The term cell tissue comprises completeorgans or parts of organs of an animal, however also specific cell linesthat can be isolated and cultivated therefrom, i.e. increased in number.

[0002] A recombinant DNA expression vector is an instrument forproducing a transgenic animal carrying, among other features, theforeign DNA to be integrated in the cells of the animal.

BACKGROUND OF THE INVENTION

[0003] The general technological background of the special transgenicanimal provided by the invention is the following. Cancer, in particularlung cancer, is one of the most widespread diseases of mankind, and hasup to now therapy predictions offering little success only. In theframework of the development of better therapies for cancer diseases itis, among other conditions, required and also legally laid down, forethical reasons, to perform pre-clinical examinations in animal modelswith possible active substances obtained from basic research or byscreening tests. In the case of examinations of prospective activesubstances for cancer therapies, it is therefore required to provideanimals or (animal) cell tissues having the respective cancer diseasesto be investigated, in order that the physiological effects, possiblyalso side effects, of the active substances can be tested in aqualitative and quantitative manner.

[0004] Cancer diseases are caused in many cases by the effects ofso-called oncoproteins. These are proteins that have differentstructures compared to corresponding proteins in a healthy organism.These oncoproteins are capable, through a not yet fully understoodprocesses, to transform normal cells into uncontrollably proliferatingcells, i.e. cancer cells. The formation of oncoproteins in an organismis in turn caused by so-called oncogenes, i.e. genes coding for theoncoprotein. Oncogenes may be introduced into a cell by viruses, mayhowever also be formed by way of mutation of (certain) “healthy” genes,the proto-oncogenes. Such mutations can for instance take place bytranslocation (displacement) of a gene responsible for the production ofa protein within the genome, by point mutations (replacement of a baseand/or individual bases in the DNA of a gene responsible for theproduction of a protein by a different base, with the consequence of theformation of a protein of modified amino acid sequence, the oncogene),by deletion (removal of one or more bases) or also by mutations in theregion of a so-called promoter applicable for the respective gene. As apromoter is designated a DNA region of a gene by means of which thetranscription (of the DNA code into a corresponding RNA) and thusfinally also the expression (formation) of the protein correlated withthe gene can be controlled. In a natural manner, a specific promoter isusually assigned to each gene, this promoter being arranged ahead of thelatter in the genome. Ahead means that the promoter in the DNA sequencehas a certain distance to the starting point of a transcription. Forinitiating a transcription, it is then also required that so-calledtranscription factors (often specific for the cell type) are taken up bythe promoter.

[0005] In particular in connection with lung cancer, the so-called Rafproto-oncogenes play a special role. These genes are highly conservativewith regard to evolution, and code kinases specific for serine/threonineof the cytoplasm playing in turn a role in the mitogenic signaltransduction. Known in the art are for instance the genes A, B andc-Raf-1. For a survey, reference is made to documents U. R. Rapp et al.,The Oncogene Handbook, Elsevier Science Publishers, Netherlands, page115-154, 1988, and U. R. Rapp, Oncogene, 6, 495, 1991. To the family ofthe Raf genes belongs, among others, the c-Raf-1 gene expressing thec-Raf-1 kinase ubiquitarily in an organism. The c-Raf-1 gene comprisesthree conserved regions, i.e. these regions are in accordance withcorresponding regions of other Raf genes of the family. The region CR1is a regulatory domain around a cys finger consensus sequence, theregion CR2 is a region having a high content of serine or threonine, andCR3 is the kinase domain. With regard to further detailed information,reference is made to document U.S. Pat. No. 5,618,670. From thisdocument are also known (partial) sequences of the natural form of theCR3 region of the c-Raf-I gene of mice and (partial) sequences ofvarious point mutations thereof From document U.S. Pat. No. 5,156,841are known plasmids and eucariotic expression vectors containing A-Rafand v-Raf oncogenes, however in different connections, namely the genicproduction of Raf oncoproteins for immunological investigations.

DETAILED DESCRIPTION OF THE INVENTION

[0006] The invention is based on the technical problem to providenon-human mammals in sufficiently high numbers and with a pathologybeing uniform and reproducible with- regard to tumor formation, for thepurpose of pre-clinic examinations of prospective anti-cancer substancesor therapies.

[0007] For achieving this object, the invention teaches a transgenicnon-human mammal whose cells express a constitutively active oncogenicmutant of the kinase-domain ofthe c-Raf-1 gene or a protein coded by acorresponding normal allele or a derivative of the A, B or c-Raf-1 gene.—The term constitutively active means, in the context of the invention,that the protein per se is always active, i.e. the physiological effectof the protein is always obtained even without the condition of furtherreaction cascades in a cell or an organism. In contrast thereto, theactivation of the not constitutively active Raf-1 protein requires forinstance the bonding of the Raf protein with the Raf-1 protein. The termconstitutively active therefore refers, for the purpose of theinvention, only to the protein or the corresponding gene code and not tothe gene itself or the gene activation. The reference to the Raf-1 genemeans Raf-1 genes or variants thereof existing in any organism, at leasthowever such Raf-1 genes existing in mammals. By the fact that themammal is a transgenic animal having the mentioned features, identicalanimals, with regard to the pathology of the tumors induced by theexpression of the mutant, can be obtained in any number by way of thenatural reproduction from a transgenic base animal. Thereby pre-clinicexaminations of active substances or therapies can be performed with therequired reproducibility and the required statistical significance, andthat also with defined control groups.

[0008] For performing pre-clinic examinations of active substances andtherapies against lung cancer it is recommended that the expression ofthe protein coded by the constitutively active oncogenic mutant of thekinase-domain of the c-Raf-1 gene or by a corresponding allele or aderivative of the A, B or c-Raf-1 gene takes place in lung cells, sincethen the animals develop reproducible lung tumors.

[0009] In structural regard, a mammal as described above ischaracterized by that it contains foreign DNA with a constitutivelyactive oncogenic mutant of the kinase-domain of the c-Raf-1 gene or witha corresponding normal allele or a derivative of the A, B or c-Raf-1gene. Alleles or derivatives are variants in a DNA sequence virtuallynot affecting the basic function of the respective gene.

[0010] Advantageously, the foreign DNA in addition contains a promoterfor the surfactant protein C, preferably for the human surfactantprotein C, and this promoter is arranged in the foreign DNA with theproviso that by the promoter the transcription of the mutant of thekinase-domain of the c-Raf-1 gene or of a corresponding normal allele ora derivative of the A, B or c-Raf-1 gene is controlled in other words,the promoter being in a natural manner arranged ahead of the gene codingthe surfactant protein C, is arranged instead, according to theinvention, at a suitable position of the mutant or of the gene. Theprecise arrangement of promoter and mutant or gene with regard to eachother is usual knowledge of the man skilled in the art. If the exactpositioning of the speciai promoter ahead of the gene utilized accordingto the invention cannot be derived from basic considerations, simpletests with different variants of positioning can however be performed,in order to determine a suitable position: The number of variants inquestion, under consideration of the general technical knowledge, ishowever very limited. The surfactant protein C plays a role for thesurfactant factor reducing the alveolar surface tension between the lungepithelium and air and thus preventing that the alveoli will collapseduring breathing-out and that the epithelia will stick together. Byapplication of the promoter for the surfactant protein C it is achievedthat only the transcription factors specifically or with increasedfrequency occurring in the lung and inter-reacting with this promotercan so to speak switch on the mutant, with the result that lung tumorswill be formed at high selectivity and reproducibility.

[0011] In a preferred further embodiment of the invention, the foreignDNA in addition contains DNA of the SV40 virus. SV means Simian Virus.This comprises the polyadenylated sequence and intron/exon regions ofthe SV40 virus being known in the art (see description of the followingFIG. 1). The integration of this SV40 DNA causes an increase of thetranslation efficiency of the polyadenylated mRNA. In detail it ispreferred that the mammal comes from the group of rodents.

[0012] A transgenic non-human mammal as described above is obtainable bythe following steps: a) integration of the cDNA sequence of aconstitutively active oncogenic mutant of the kinase-domain of thec-Raf-1 gene or of a corresponding normal allele or a derivative of theA, B or c-Raf-1 gene in an expression vector, b) insertion of thetransgenic vector obtained in step a), preferably after linearization,in pronuclei of fertilized oocytes from a non-human mammal, c)implantation of the oocytes obtained in step b) in brood animals of thesame species as the donor species of the oocytes and delivery ofdescendant animals from the oocytes, d) genotypization and selection ofthe descendant animals obtained in step c) with the proviso that cellsof the selected mammals express a constitutively active oncogenic mutantof the kinase-domain of the c-Raf-1 gene or a protein coded by acorresponding normal allele or a derivative of the A, B or c-Raf-1 gene.Genotypization can be obtained by means of methods well known to the manskilled in the art, for instance by tail biopsy by means of PCR(polymerase chain reaction, a method for the in-vitro amplification of adefined DNA fragment) and Southern Blot (a method for the analysis ofDNA fragments in DNA preparations), with those mammals being selectedwhose cells can be proven by the examinations of the genotypization tocontain the foreign DNA according to the invention. Advantageously, theexpression vector used is step a) contains a promoter for the surfactantprotein C, preferably a promoter for the human surfactant protein C.This promoter is arranged in the foreign DNA with the proviso that bythe promoter the transcription of the mutant of the kinase-domain of theRaf-1 gene or of a corresponding normal allele or derivative of the A, Ror c-Raf-1 gene is controlled. In detail, the foreign DNA may comprisefor instance either the healthy (FIG. 1) or a constitutively activeoncogenic mutant of the Raf-1 gene with a sequence according to one ofFIG. 1, however with deletion as ΔRaf(26-302). In place of a deletion,point mutations of the sequence shown in FIG. 1 can also be used. Suchpoint mutations can for instance specifically be caused byadministration of 1-ethyl-1-nitrosourea (ENU) to animals having thehealthy sequence, thereby such mutants being accessible in a simplemanner.

[0013] The invention also relates to a method for producing a transgenicnon-human mammal as described above according to patent claims 10 and11. Non-human mammals according to the invention are used for pre-clinicexaminations of the effectiveness of substances directed against lungcarcinomas and/or therapeutical methods directed against lungcarcinomas, in particular for the pre-clinic examination of theeffectiveness of substances inhibiting Raf-kinase. Such substancescompletely inhibit or reduce the activity of Raf-kinases, therebypossibly a means for the deactivation particularly of Raf-oncoproteinsand therefore for the proliferation inhibition of tumor cells beingfound. Another advantageous utilization of a non-human, mammal accordingto the invention is the investigation of the pathogenesis of lungtumors, thereby a better understanding of the disease per se beingpossible.

[0014] The invention however also relates to cell tissues, in particularlung tissues, from a transgenic non-human mammal according to one ofclaims 1 to 9, which cell tissue has a higher probability of tumorformation, preferably of lung tumors, to a method for the productionthereof according to claims 16 and 17 and to the utilization thereofaccording to claims 18 to 20. With regard to the cell tissue or alsospecific cell lines comprised therein, isolated and possibly cultivatedtherefrom, all general explanations given above apply in correspondingmanner.

[0015] Finally the invention also comprises a recombinant DNA expressionvector containing A) the DNA sequence of a constitutively activeoncogenic mutant of the kinase-domain of the c-Raf-1 gene or of acorresponding normal allele or a derivative of the A, B or c-Raf-1 gene,B) a promoter domain for the surfactant protein C, by means of which thetranscription of the DNA sequence defined in A) is controllable, C) asan option the DNA sequence of the SV40 virus. By means of such a vectorthe transgenic mammals according to the invention and possibly also thecell tissues therefrom can be produced. As an example, the DNA sequencedefined in A) comprises a sequence according to FIG. 4 or to the end ofthe specification, respectively, or a sequence ΔRaf(26-302) derivedtherefrom, and/or the promoter domain defined in B) is a promoter domainfor the human surfactant protein C. The reproduced sequence is that ofhuman-c-Raf-1. Instead, also the c-Raf-1 sequences of the mouse, wildtype or mutated, according to document 25 U.S. Pat. No. 5,618,670 can beused. Other sequences, even from other organisms, are also possible, asfar as they are basically a Raf-1 sequence.

[0016] Subject matter of the invention is further a screening methodwith utilization of the transgenic non-human mammals according to theinvention or of cell tissues therefrom, a group of prospective activesubstances against cancer, in particular lung cancer, being administeredto the animals, and an evaluation of the effects of each individualprospective active substance with regard to proliferation inhibition,oncoprotein inhibition or the like is performed. The invention furthercomprises active substances that can be detected by such a screeningmethod as being sufficiently effective.

[0017] Plasmids with transgenic vectors according to the invention(active oncogenic mutant of the kinase-domain of the human c-Raf-1 gene,i.e—plasmid SPC-ΔRaf(26-302) and a transgenic vector with normal humanc-Raf-1 gene, i.e. plasmid SPC-Raf-1) were registered at DSMZ-DeutscheSammlung von Mikroorganismen and Zellkulturen GmbH, Mascheroder Weg 1b,D-38124 Braunschweig. The registration number for the plasmid SPC-ΔRaf(26-302) is 11849. Thus the invention also relates to transgenic vectorsas registered, as well as to transgenic animals or cells or cell tissuesto be produced therefrom, and to the utilizations described above ofsuch animals or cells or cell tissues.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In the following, the invention will be described by figures andembodiments representing examples only. There are:

[0019]FIG. 1 is a schematic representation of the structure of vectorssuitable for the invention,

[0020]FIG. 2 is a Southern Blot of selected transgenic animals accordingto the invention,

[0021]FIGS. 3a-3 b are Western or Northern Blots, respectively, ofobtained animal lines according to the invention, and

[0022]FIG. 4 is the DNA sequence (linear) of the kinase-domain of thehuman-c-Raf-1 gene.

EXAMPLES [HUMAN Raf]

[0023] The general procedure during the production of transgenic miceaccording to the invention was the following. The cDNA sequences of thetransforming c-Raf subdomain (Raf B×B) were cloned into a lung-specificexpression vector containing the promoter region of the humansurfactant-associated protein C (SPC). After restriction digestion andlinearization of these transgenic vectors schematically shown in FIG. 2,the respective foreign DNA was inserted into the pronuclei of fertilizedoocytes, then implanted into brood mice. The descendants of such micewere genotyped by tail end biopsy by means of PCR and Southern Blot. Bythe thus identified founder animals, mouse lines were established, inwhose lungs the expression of the transgene was detected in Western orNorthern Blot or by means of RT-PCR.

[0024]FIG. 1 shows lung-directed expression vectors for the proteinkinase Raf-1. The transgenic vectors include the 3.7 kb (kilobases) wideregion of the human SPC promoter, the 3.0 kb c-Raf-1 or 1.4 kb humanΔRaf (26-302) cDNA 10 fragments and a 0.4 kb fragment of viral DNAincluding the polyadenylation sequence and intron/exon regions of theSV40 virus, thereby the translation efficiency of the polyadenylatedmRNA being increased. Further are drawn the intersections of the used(also in the following examples) restriction endonucleases.

[0025] The individual steps of the exemplary general procedure describedabove are explained with regard to the experimental measures in detailin the following examples.

Example 1

[0026] Cloning of the cDNA sequences of the transforming c-Raf subdomaininto a lung-specific expression vector was performed as follows. Thevector SPC-Raf-1 for generating the wild type-Raf-1 transgenic mice wasproduced by that a 3.0 kb fragment of the human Raf-1 cDNA (Bonner, T.I.; Oppermann, R.; Seebrug, P.; Kerby, S. B.; Gunnell, M. A.; Young, A.C.; and Rapp, U. R.; 1986; “The complete coding sequence of the humanraf oncogene and the corresponding structure of the c-raf-1 gen.”;Nucleic Acids Res.; 14, 109) was cloned in the EcoRI interface of theplasmid SPC3.7/5V40 including the 3.7 kb promoter region of the humansurfactant-associated protein C (SPC) (Korfhagen, T. R.; Glasser, S. W.,Wert, S. E., Bruno, M. D.; Daugherty, C. C.; McNeish, J. D.; Stock, J.L.; Potter, S. S.; Whitsett, J. A.; 1990; “Cis-acting sequences from ahuman surfactant protein gene confer pulmonary-specific gene expressionin transgenic mice.”; Proc. Natl. Acad. Sci.; 87, 6122). In analogusmanner, the transgenic vector SPC-ΔRaf (26-302) was cloned by insertionof a 1.4 kb fragment, of human Raf-1 cDNA that has been obtained bydeletion of the amino acids 26 to 302 of the regulatory domain (Bruder,J. T.; Heidecker, G.; and Rapp, U. R.; 1992; “Serum-, TPA-, andRasinduced expression from Ap-1/Ets-driven promoters requires Raf-1kinase.”; Genes and Dev.; 6, 545) and thus contains the kinase-domain ofthe Raf-1 protein (activated oncogenic mutant of the Raf-1 kinase).

Example 2

[0027] The linearization and the pronucleus injection were performed asfollows. The transgenic vectors were cut with the restrictionendonucleases NotI and NdeI, cleaned with a preparative agarose gel(Sambrooks et al., 1989, see below), and diluted to a concentration of 1ng/ml. 200 ng of the linearized DNA fragments were injected into themale pronuclei of fertilized oocytes. Transgenic founder mice wereidentified by analysis of the genomic DNA isolated from tail ends bySouthern Blot (see also example 3). The founder mice were crossed withnon-transgenic B6D2 mice, in order to establish stable lines.

[0028] The used mice were C57BL/6×DBA F2 mice (B6D2 mice), and wereobtained from Harlan Winkelmann (Borchen) and from Charles River(Sulzfeld), and were held and bred on in the stable of the MSZ (Institutfür Medizinische Strahlenkunde and Zellforschung, Würzburg University,097078 würzburg) under pathogen-free conditions.

Example 3

[0029] PCR and Southern Blot for genotypization were performed by thefollowing operational instructions. For the detection of the transgenicintegration, 10 μg genomic DNA from tail ends were cut over night with40 units BamHI, separated on a 0.7 % agarose gel by means ofelectrophoresis, and transferred by means of a capillary blot tonitrocellulose (Sambrook, J.; Fritsch, E. F.; Maniatis, T.; “MolecularCloning: a laboratory manual)”; Cold Spring Harbor Laboratory Press).After fixation of the DNA by u.v. light followed the detection of thetransgene by hybridization of the membrane (Church, G. M. and Gilbert,W.; “Genomic sequencing”; Proc. Natl. Acad. Sci.; 81, 1991) with a Raf-1probe (containing the Raf-1/SV40 sequence) that was obtained bydigestion of the transgenic vector SPC-Raf-1 with BamHI. The bound probecross-hybridizing also with the mouse-Raf locus, was detected byexposure of the membrane on film material, and marked a 3.4 kb Raf1/SV40 fragment or a 1.8 kb ΔRaf(26-302)/SV40 fragment, respectively.The result of Southern Blots from selected animals according to theinvention is shown in FIG. 2. Therein the positions of endogenic Raf-1and of the transgenic ΔRaf (26-302) or Raf-1 fragments in transgenic (T)and non-transgenic (NT) mice for two independent mouse lines (ΔRaf_(—)11and ΔRaf_(—)23 or Raf 74 and Raf 87, respectively) can be seen. Thepositions of the respective fragments are identified by arrows. It caneasily be found that the bands to be assigned to transgenic ΔRaf(26-302)or Raf-1 fragments, respectively (lower arrows), are only registered forthe transgenic animals.

Example 4

[0030] In total 2 c-Raf-B×B(or ΔRaf(26-302), respectively) mouse lineswere obtained. These mice according to the invention of both linesdeveloped at an age of 6 to 7 months massive lung carcinomas, thecellular origin of which corresponded with regard to distributionpattern and pheno type to the pneumocytes type II. Investigations of thetime dependence of the tumor development showed that after 2 monthsalready significant multi-centric tumor formation occurred. In the lungtissue of both lines could also be detected the expression of thetransgene in Western Blot, as visible in FIG. 3. Thus also thecorrelation between specific misapplied Raf kinase activity and lungtumor induction is proven in vivo.

[0031] The Western Plot of FIG. 3 was obtained as follows. The proteinexpression of the transgene was detected by that lung tissue of the micewas lysated in detergent containing buffer, and approx. 100 μg of thesolubilized proteins were split with a 10 % SDS polyacrylamid gel. Aftertransfer of the proteins to nitrocellulose, the membrane was incubatedover night with milk-powder containing blocking solution andsuccessively for 1 hour each with a polyclonal anti-Raf-I rabbitanti-serum and with peroxidace-coupled anti-rabbit immunoglobulin. Aftereach incubation step, several times washing solution was employed. Thedetection of the bound anti-bodies took place by the reaction of theperoxidase with a chemiluminescence emitting substrate and exposure onfilm material. In FIG. 3 is shown in detail an immuno blot withsolubilized proteins from lung tissue of the specified mouse lines andfrom non-transgenic control mice (C) afterstaining with the anti-Rafrabbit anti-serum. Specified are the positions of the respective Rafproteins at approx. 74 kDa (Raf-1) and 42 kDa (ΔRaf(26-302)). In FIG. 3ais identified the endogenic Raf-1 of the mouse and the ΔRaf(26-302) inthe transgenic mouse lines. In FIG. 3b can be seen, due to the identicalmolecular weight of endogenic murine Raf-1 and transgenic human Raf-1,the transgenic expression caused by the increase in intensity of theRaf-1 band.

[0032] The sequence listing in FIG. 4 is repeated in the following.

1 1 1 2977 DNA Human exon (105)..(105) exon (337)..(337) exon(451)..(451) exon (553)..(553) exon (712)..(712) exon (811)..(811) exon(963)..(963) exon (994)..(994) exon (1120)..(1120) exon (1237)..(1237)exon (1324)..(1324) exon (1501)..(1501) exon (1549)..(1549) exon(1667)..(1667) exon (1798)..(1798) exon (1933)..(1933) 1 ccgaatgtgaccgcctcccg ctccctcacc cgccgcgggg aggaggagcg ggcgagaagc 60 tgccgccgaacgacaggacg ttggggcggc ctggctccct cagg t ttaagaattg 115 tttaagctgcatcaatggag cacatacagg gagcttggaa gacgatcagc aatggttttg 175 gattcaaagatgccgtgttt gatggctcca gctgcatctc tcctacaata gttcagcagt 235 ttggctatcagcgccgggca tcagatgatg gcaaactcac agatccttct aagacaagca 295 acactatccgtgttttcttg ccgaacaagc aaagaacagt g g tcaatgtgcg 347 aaatggaatgagcttgcatg actgccttat gaaagcactc aaggtgaggg gcctgcaacc 407 agagtgctgtgcagtgttca gacttctcca cgaacacaaa ggt a aaaaagcacg 461 cttagattggaatactgatg ctgcgtcttt gattggagaa gaacttcaag tagatttcct 521 ggatcatgttcccctcacaa cacacaactt t g ctcggaagac gttcctgaag 573 cttgccttctgtgacatctg tcagaaattc ctgctcaatg gatttcgatg tcagacttgt 633 ggctacaaatttcatgagca ctgtagcacc aaagtaccta ctatgtgtgt ggactggagt 693 aacatcagacaactctta t tgtttccaaa ttccactatt ggtgatagtg gagtcccagc 752 actaccttctttgactatgc gtcgtatgcg agagtctgtt tccaggatgc ctgttagt t 811 ctcagcacagatattctaca cctcacgcct tcacctttaa cacctccagt ccctcatctg 871 aaggttccctctcccagagg cagaggtcga catccacacc taatgtccac atggtcagca 931 ccacgctgcctgtggacagc aggatgattg a g gatgcaattc gaagtcacag 983 cgaatcagcc tcaccttcagc cctgtccagt agccccaaca atctgagccc 1034 aacaggctgg tcacagccgaaaacccccgt gccagcacaa agagagcggg caccagtatc 1094 tgggacccag gagaaaaacaaaatt a ggcctcgtgg acagagagat tcaagctatt 1150 attgggaaat agaagccagtgaagtgatgc tgtccactcg gattgggtca ggctcttttg 1210 gaactgttta taagggtaaatggcac g gagatgttgc agtaaagatc ctaaaggttg 1267 tcgacccaac cccagagcaattccaggcct tcaggaatga ggtggctgtt ctgcgc a 1324 aaacacggca tgtgaacattctgcttttca tggggtacat gacaaaggac aacctggcaa 1384 ttgtgaccca gtggtgcgagggcagcagcc tctacaaaca cctgcatgtc caggagacca 1444 agtttcagat gttccagctaattgacattg cccggcagac ggctcaggga atggac t 1501 atttgcatgc aaagaacatcatccatagag acatgaaatc caacaat a tatttctcca 1559 tgaaggctta acagtgaaaattggagattt tggtttggca acagtaaagt cacgctggag 1619 tggttctcag caggttgaacaacctactgg ctctgtcctc tggatgg c cccagaggtg 1677 atccgaatgc aggataacaacccattcagt ttccagtcgg atgtctactc ctatggcatc 1737 gtattgtatg aactgatgacgggggagctt ccttattctc acatcaacaa ccgagatcag 1797 a tcatcttcat ggtgggccgaggatatgcct ccccagatct tagtaagcta 1848 tataagaact gccccaaagc aatgaagaggctggtagctg actgtgtgaa gaaagtaaag 1908 gaagagaggc ctctttttcc ccag atcctgtcttc cattgagctg ctccaacact 1963 ctctaccgaa gatcaaccgg agcgcttccgagccatcctt gcatcgggca gcccacactg 2023 aggatatcaa tgcttgcacg ctgaccacgtccccgaggct gcctgtcttc tagttgactt 2083 tgcacctgtc ttcaggctgc caggggaggaggagaagcca gcaggcacca cttttctgct 2143 ccctttctcc agaggcagaa cacatgttttcagagaagct ctgctaagga ccttctagac 2203 tgctcacagg gccttaactt catgttgccttcttttctat ccctttgggc cctgggagaa 2263 ggaagccatt tgcagtgctg gtgtgtcctgctccctcccc acattcccca tgctcaaggc 2323 ccagccttct gtagatgcgc aagtggatgttgatggtagt acaaaaagca ggggcccagc 2383 cccagctgtt ggctacatga gtatttagaggaagtaaggt agcaggcagt ccagccctga 2443 tgtggagaca catgggattt tggaaatcagcttctggagg aatgcatgtc acaggcggga 2503 ctttcttcag agagtggtgc agcgccagacattttgcaca taaggcacca aacagcccag 2563 gactgccgag actctggccg cccgaaggagcctgctttgg tactatggaa cttttcttag 2623 gggacacgtc ctcctttcac agcttctaaggtgtccagtg cattgggatg gttttccagg 2683 caaggcactc ggccaatccg catctcagccctctcaggag cagtcttcca tcatgctgaa 2743 ttttgtcttc caggagctgc ccctatggggcgggccgcag ggccagcctg tttctctaac 2803 aaacaaacaa acaaacagcc ttgtttctctagtcacatca tgtgtataca aggaagccag 2863 gaatacaggt tttcttgatg atttgggttttaattttgtt tttattgcac ctgacaaaat 2923 acagttatct gatggtccct caattatgttattttaataa aataaattaa attt 2977

What is claimed is:
 1. A transgenic mouse whose cells express aconstitutively active oncogenic mutant of the kinase domain of thec-Raf-1 gene or a protein coded by a corresponding normal allele of theRaf gene or a derivative of the A Raf gene, the B Raf gene or thec-Raf-1 gene.
 2. The transgenic mouse according to claim 1, wherein theexpression takes place in lung cells.
 3. A transgenic mouse, wherein themouse contains a foreign DNA having a constitutively active oncogenicmutant of the kinase-domain of the c-Raf-1 gene or with a correspondingnormal allele of the Raf gene or a derivative of the A Raf gene, the BRaf gene or the c-Raf-1 gene.
 4. The transgenic mouse according to claim3, wherein the foreign DNA in addition contains a promoter for thesurfactant protein C and wherein this promoter is arranged in theforeign DNA with the proviso that by the promoter the transcription ofthe mutant of the kinase-domain of the c-Raf-1 gene or of acorresponding normal allele of the Raf gene or a derivative of the A Rafgene, the B Raf gene or the c-Raf-1 gene is controlled.
 5. Thetransgenic mouse according to claim 3, wherein the foreign DNA inaddition contains DNA of the SV40 virus.
 6. A transgenic mouse, which isobtainable by the following steps: a) integration of the cDNA sequenceof a constitutively active oncogenic mutant of the kinase domain of thec-Raf-1 gene or of a corresponding normal allele of the Raf gene or aderivative of the A Raf gene, the B Raf gene or the c-Raf-1 gene in anexpression vector to provide an expression vector carrying a foreignDNA, b) insertion of the transgenic vector obtained in step a) inpronuclei of fertilized oocytes from a mouse, c) implantation of theoocytes obtained in step b) in brood animals of the same species as thedonor species of the oocytes and delivery of descendant animals from theoocytes, d) genotypization and selection of the descendant animalsobtained in step c) with the proviso that cells of the selected miceexpress a constitutively active oncogenic mutant of the kinase domain ofthe c-Raf-1 gene or a protein coded by a corresponding normal allele ofthe Raf gene or a derivative of the A Raf gene, the B Raf gene or thec-Raf-1 gene.
 7. The transgenic mouse according to claim 6, wherein theexpression vector used in step a) contains a promoter for the surfactantprotein C and wherein this promoter is arranged in the foreign DNA withthe proviso that by the promoter the transcription of the mutant of thekinase-domain of the c-Raf-1 gene or of a corresponding normal allele ofthe Raf gene or a derivative of the A, B or c-Raf-1 gene is controlled.8. The transgenic mouse according to claim 3, wherein the constitutivelyactive oncogenic mutant of the c-Raf-1 gene comprises a sequenceaccording to FIG. 1 or a sequence ΔRaf(26-302) derived therefrom.
 9. Amethod for producing a transgenic mouse, including the following steps:a) integration of the cDNA sequence of a constitutively active oncogenicmutant of the kinase domain of the c-Raf-1 gene or of a correspondingnormal allele of the Raf gene or a derivative of the A Raf gene, the BRaf gene or the c-Raf-1 gene in an expression vector to provide anexpression vector carrying a foreign DNA, b) insertion of the transgenicvector obtained in step a) in pronuclei of fertilized oocytes from amouse, c) implantation of the oocytes obtained in step b) in broodanimals of the same species as the donor species of the oocytes anddelivery of descendant animals from the oocytes, d) genotypization andselection of the descendant animals obtained in step c) with the provisothat cells of the selected mice express a constitutively activeoncogenic mutant of the kinase domain of the c-Raf-1 gene or a proteincoded by a corresponding normal allele of the Raf gene or a derivativeof the A Raf gene, the B Raf gene or the c-Raf-1 gene.
 10. The methodaccording to claim 9, wherein the expression vector used in step a)contains a promoter for the surfactant protein C and wherein thispromoter is arranged in the foreign DNA with the proviso that by thepromoter the transcription of the mutant of the kinase-domain of thec-Raf-1 gene or of a corresponding normal allele of the Raf gene or aderivative of the A; B or c-Raf-1 gene is controlled.
 11. Theutilization of a mouse according to claim 1 for the pre-clinicexamination of the effectiveness of substances intended against lungcarcinomas and/or of therapeutical methods intended against lungcarcinomas.
 12. The utilization according to claim 11 for the preclinicexamination of the effectiveness of substances inhibiting Rat-kinase,13. A lung cell tissue from a transgenic mouse according claim 1, whichcell tissue has a higher probability of forming lung tumors.
 14. Amethod for producing cell tissue from a transgenic mouse, including thefollowing steps: a) integration of the cDNA sequence of a constitutivelyactive oncogenic mutant of the kinase domain of the c-Raf-1 gene or of acorresponding normal allele of the Raf gene or a derivative of the A Rafgene, the B Raf gene or the c-Raf-1 gene in an expression vector toprovide an expression vector carrying a foreign DNA, b) insertion of thetransgenic vector obtained in step a) in pronuclei of fertilized oocytesfrom a mouse, c) implantation of the oocytes obtained in step b) inbrood animals of the same species as the donor species of the oocytesand delivery of descendant animals from the oocytes, d) genotypizationand selection of the descendant animals obtained in step c) with theproviso that cells of the selected mice express a constitutively activeoncogenic mutant of the kinase domain of the c-Raf-1 gene or a proteincoded by a corresponding normal allele of the Raf gene or a derivativeof the A Raf gene, the B Raf gene or the c-Raf-1 gene, e) removal ofcell tissue from the mouse.
 15. The method according to claim 14,wherein the expression vector used in step a) contains a promoter forthe surfactant protein C and wherein this promoter is arranged in theforeign DNA with the proviso that by the promoter the transcription ofthe mutant of the kinase-domain of the c-Raf-1 gene or of acorresponding normal allele of the Raf gene or a derivative of the A Rafgene, the B Raf gene or the c-Raf-1 gene is controlled.
 16. Theutilization of a cell tissue according to claim 13 for the pre-clinicexamination of the effectiveness of substances intended against lungcarcinomas and/or of therapeutical methods intended against lungcarcinomas.
 17. The utilization according to claim 16 for the preclinicexamination of the effectiveness of substances inhibiting Raf-kinase.18. The transgenic mouse according to claim 4, wherein the promoter forthe surfactant protein C is for the human surfactant protein C.
 19. Thetransgenic mouse according to claim 6, wherein insertion of thetransgenic vector obtained in step occurs after linearization.
 20. Thetransgenic mouse according to claim 6, wherein the promoter for thesurfactant protein C is for the human surfactant protein C.
 21. Themethod according to claim 9, wherein the promoter for the surfactantprotein C is for the human surfactant protein C.
 22. The methodaccording to claim 9, wherein the expression vector used in step a) inaddition contains DNA of the SV40 virus.
 23. The method according toclaim 14, further comprising cultivation of the removed cell tissue. 24.The utilization of a non-human mammal according to claim 1 for theexamination of the pathogenesis of lung tumors.
 25. The utilization of acell tissue according to claim 13 for the examination of thepathogenesis of lung tumors.
 26. A recombinant DNA expression vectorcontaining a) the DNA sequence of a constitutively active oncogenicmutant of the kinase-domain of the c-Raf-1 gene or of a correspondingnormal allele or a derivative of the A, B or c-Raf-1 gene, b) a promoterdomain for the surfactant protein C, by means of which the transcriptionof the DNA sequence defined in a) is controllable, c) as an option theDNA sequence of the SV 40 virus.
 27. A recombinant DNA expression vectoraccording to claim 26, wherein the DNA sequence defined in a) is asequence according to FIG. 1 or a sequence ΔRaf (26-302) derivedtherefrom, and/or wherein the promoter domain defined in b) is apromoter domain for the human surfactant protein c.
 28. The utilizationof a recombinant DNA expression vector according to claim 26 forproducing a transgenic mammal or a cell tissue from such a mammal.